Thrombin is a serine protease that facilitates blood clotting by catalyzing the conversion of fibrinogen to fibrin. Thrombin is also responsible for activating platelets and indirectly responsible for regulation of its own production and inhibition through multiple proteolytic feedback mechanisms. Thrombin is also involved in activation of factor VIII, factor V, factor XI, factor XIII and protein C. Thrombin is widely used in clinical applications as a coagulation factor to staunch bleeding of wounds by conversion of fibrinogen to fibrin. Thrombin is a common component of surgical dressings, and has been used in combination with fibrinogen and other coagulation proteins in two-component hemostatic systems such as fibrin glues, adhesives, and sealants.
Thrombin is produced by proteolytic activation of the precursor (zymogen) prothrombin. For the production of thrombin, prothrombin must be cleaved at two sites generating intermediate products. The conversion of prothrombin to thrombin in the body is catalyzed by the prothrombinase complex which includes activated Factor X and Factor V and assembles on negatively charged phospholipid membranes in the presence of calcium ions.
Thrombin may be manufactured from prothrombin by contacting a source of prothrombin (such as blood plasma or a blood fraction), with a solid adsorbent capable of adsorbing the prothrombin from the source of prothrombin, for example barium sulfate (BaSO4). The solid adsorbent is typically washed using a washing solution to remove contaminants such as unbound proteins, and subsequently the prothrombin is eluted therefrom using an elution solution. Subsequent to additional optional purification and processing steps, the eluted prothrombin can be converted to thrombin by activation using an activator, e.g., calcium ions. It has been reported that significant differences in yield of thrombin from a given volume of a source of prothrombin are found when using different BaSO4 reagents, such as BaSO4 reagents obtained from different manufacturers or even different batches of reagent produced by a specific manufacturer.
It has been proposed in reports that differences in thrombin yield are at least partially attributable to variations in adsorption of prothrombin by different BaSO4 reagents and that adsorption capacity is a key factor in the choice of BaSO4 reagent for prothrombin adsorption. Surgenor and Neortker (1952) stated that certain BaSO4 reagents are more effective than others in adsorption of prothrombin from plasma while Voss D. (1965) noted that when using different BaSO4 reagents for adsorption of prothrombin complex, significant differences were found in the amount of BaSO4 needed to adsorb the same amount of prothrombin complex and speculated that this was due to differences in their crystal structure. However, in the Applicant's case, the morphology of different batches of BaSO4 have been tested and no significant differences were determined. Also some ions, e.g. Ca2+, were reported to contribute to different adsorption rates for different BaSO4 batches, and in this case, the presence of Ca2+ in different batches of BaSO4 was not significantly different either.
Thrombin manufacturers attempt to increase thrombin yield by resorting to a trial and error method of selecting a suitable BaSO4 reagent from among multiple different BaSO4 reagents.